The vector sequence has been compiled using the information from sequence databases, published literature, and other sources, together with partial sequences obtained by Evrogen. This vector has not been completely sequenced.
pmKate2-clathrin is a mammalian expression vector encoding mKate2-clathrin fusion protein (see reporter description). The vector can be used for fluorescent labeling of clathrin LCB in living cells.
C-terminal mKate2 fusion construct with human clathrin light chain expressed in human cervical adenocarcinoma cells (HeLa).
Scale bar represents 10 μm. Image from Shcherbo et al., 2009.
mKate2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Human clathrin LCB is fused to the mKate2 C-terminus. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the mKate2-clathrin coding sequence [Kozak, 1987].
pmKate2-clathrin vector can be used as a source of mKate2-clathrin hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.
Note: The plasmid DNA was isolated from dam+-methylated
The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in
SV40 early promoter (PSV40) provides neomycin resistance gene (Neor) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr) in
Expression in mammalian cells
pmKate2-clathrin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the mKate2-clathrin fusion in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].
Suitable host strains for propagation in
Location of features
PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
Kozak consensus translation initiation site: 606-616
mKate2-clathrin fusion: 613-1992
Start codon (ATG): 613-615
Last amino acid in mKate2: 1312-1314
Clathrin LCB: 1360-1992
Stop codon: 1993-1995
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2156-2161 & 2185-2190
mRNA 3' ends: 2194 & 2206
f1 single-strand DNA origin: 2253-2708
Bacterial promoter for expression of Kanr gene
-35 region: 2770-2775
-10 region: 2793-2798
Transcription start point: 2805
SV40 origin of replication: 3049-3184
SV40 early promoter
Enhancer (72-bp tandem repeats): 2882-2953 & 2954-3025
21-bp repeats: 3029-3049, 3050-3070 & 3072-3092
Early promoter element: 3105-3111
Major transcription start points: 3101, 3139, 3145 & 3150
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3233-3235
Stop codon: 4025-4027
G->A mutation to remove Pst I site: 3415
C->A (Arg to Ser) mutation to remove BssH II site: 3761
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4263-4268 & 4276-4281
pUC plasmid replication origin: 4612-5255
mKate2-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 7,638,615; European Pat. 1994149; and other Evrogen Patents and/or Patent applications pending. By use of these Products, you accept the terms and conditions of the applicable Limited Use Label License.
The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839, and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242.
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